SUMMARY Adeno-associated viruses (AAV) have become increasingly popular as vectors for human gene therapy. However, the requirement for high vector doses needed to achieve therapeutic efficacy poses a challenge to cost-effective manufacturing of AAV vectors for clinical use. The fundamental process of AAV vector production is virion assembly, the process by which 60 VP capsid protein subunits form an icosahedral protein shell followed by AAV vector genome packaging. Unfortunately, this basic process remains poorly understood and there is an urgent need to elucidate the process and mechanisms underlying AAV vector production. Such an understanding also could potentially uncover the key to production of high titer and high-quality AAV vectors. In this regard, there was a paradigm-shifting discovery in 2010 that the AAV cap gene expresses assembly-activating protein (AAP), a previously unidentified non-structural protein that promotes capsid assembly. Unexpectedly, studies on AAV capsid assembly from our group and others in the post-AAP discovery era have convincingly demonstrated that the AAV capsid assembly process is not conserved among different AAV serotypes, that is, knowledge of AAV capsid assembly built in the pre-AAP discovery era through studies using AAV2 is not translatable to capsid assembly of other AAV serotypes. Our preliminary data has challenged the long-standing dogma of AAV capsid assembly in the nucleolus and revealed significant serotype-dependent heterogeneity in the capsid assembly process. In addition, there is a growing appreciation for additional roles that AAP plays beyond promoting capsid assembly. Here, in order to advance our understanding of AAV vectors and improve their effective utilization in gene therapy, we seek to thoroughly understand the AAV capsid assembly process and the multifaceted roles of AAP in virion assembly of AAV vectors, using robust approaches including high-throughput mutagenesis, directed evolution, barcoding, proximity-based labeling based on BioID, and state-of-the-art bottom-up and top-down mass spectrometry. Furthermore, we will explore various potential strategies to enhance the yield and quality of AAV vectors through manipulation of various pathways we will identify in the project. Thus, the specific aims of this project are: (Aim1) To comprehensively understand AAP biology in the process of virion assembly of AAV vectors that varies across different AAV serotypes; (Aim 2) To identify host cell proteins involved in AAV capsid assembly; and (Aim 3) To explore novel strategies to enhance the yield and quality of AAV vectors by manipulating the process of virion assembly. Our project will not only address fundamental questions about the mechanism of virion assembly of AAV vectors, but also substantially further our understanding of AAV-host interactions in general. The project also has potential to discover novel strategies to improve vector production.